Use of the Ca2+ reporter GFP-aequorin for
probing infection and physiology
Inventor: Philippe BRULET et al
Description of invention:
Spatio-temporal profiles of Ca2+ signals are critical in signal transduction in living organism including animals and plants. Disruption of cellular Ca2+ homeostasis is implicated in many common diseases, including neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s as well as in schizophrenia. Several techniques, including electrophysiological recording, voltage and Ca2+ sensitive fluorescent probes allow monitoring of neuronal activation. However, none of the above is appropriate for long-term in vitro or in vivo studies. Aequorin, a genetically encoded luminescent reporter, has been used for measuring intracellular concentrations of Ca2+. It provides a very good signal above background, which makes it ideal for use in high throughput screening assays. However, the amount of light produced by aequorin is very low.
Researchers at the Institut Pasteur have developed a fluorescent/bioluminescent Ca2+ reporter, GFP-aequorin (GA) to improve the tracking, light intensity and stability of aequorin chimeras. When Ca2+ binds to aequorin, GFP emits its characteristic green light by chemoluminescent energy transfer (CRET), without a need for light excitation. Signal intensity produced by GA is several orders of magnitude higher than aequorin alone. Using fusion proteins, GA can be targeted to specific intracellular compartments, including cytosol, mitochondria, nucleus, endoplasmic reticulum (ER) and subplasmalemmal space or to a specific receptor. In a particular example, the Ca2+ reporter is under the control of the CMV promoter and can be expressed in eukaryotic cells by transient or stable transfection.
Use of the GA dual reporter should significantly improve high throughput functional screening assays of G-protein coupled receptors (GPCRs) and ion channels. In more recent work, the inventors used GA to follow in « real-time » local Ca2+ events in neural network activity. In these studies, GA was genetically targeted to different microdomains that are important in synaptic transmission. The stability of the chimeric protein makes it ideal also for expression by transgenesis. Transgenic mice expressing bioluminescent Ca2+ sensitive reporters were constructed with several targeted probes in order to visualize specific cellular events at various developmental stages. These GA transgenic mouse strains can be crossed with any mutant animals or mouse models of neurodegenerative disease to follow perturbed neural calcium homeostasis. Finally, whole animal bioluminescence calcium imaging can be performed to image specifically targeted intracellular activities and analyze various biological processes in a moving animal.
Optical imaging of cellular and subcellular calcium dynamics
Monitoring calcium fluxes or detecting electrical activity in neural cells in culture systems, organotypic cultures or living animals using non-invasive optical methods
Monitoring calcium fluxes during heart development using disease models
Study of specific molecules effects on regulation of calcium transport
Kit for detection of free calcium and diagnosis of calcium-associated disorders
High thoughpup screenin of drugs
Recent pertinent publications:
Rogers K. et al. FENS Abstr., vol.2, A079.11, 2004
Baubet V et al. Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7260-5.
US 6800492 issued October 5, 2004 and WO 01/92300 filed June 1, 2001
PCT/EP 2005/000817 filed January 11, 2005
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